Presently, malaria is a significant burden on humans. The parasite responsible for this disease continues to develop resistance to antimalarial drugs and there is no suitable vaccine to control the disease. These two major features ensure that malaria remains a major global health problem. Plasmodium falciparum causes the most severe form of the disease in humans and is responsible for 200-300 million infections per year. Greater than two million people die as a result of the disease annually. The development of an effective vaccine and the development of new and inexpensive antimalarial drugs remain top priorities in the world health community.
A key process to target for both vaccine and drug development research is the process of parasite invasion. Of the various forms of the parasite, three stages must have the ability to invade host cells: the ookinete, the sporozoite and the merozoite. There are both common features as well as unique features during the invasion process by each form. For example, although the invasive stages of the parasite are morphologically and biochemically different from one another, they share a highly conserved structural organization and special organelles called micronemes and rhopteries (for review see Sinnis and Sini, Trends in Microb. 1997 5:52-58; Pinder et al, J. Cell Sci. 2000 111:1831-1839; Chitnis, C. E. Curr. Op. in Hemat. 2001 8:85-91; and Mota and Rodriquez, Bioessays 2002 24:149-156). While function of these conserved structures has not been fully clarified, it is known that they are required for host cell invasion. All of these common structures confer some similarity to target cell invasion; the specific details in each case, however, differ. Each invasive stage of the parasite has a different target cell specificity, which is believed to be governed by a specific receptor-ligand type interaction. Furthermore, the invasion of the host cell appears to be an active process, which requires an actin-myosin based motility system to enter the host cell (Pinder et al, J. Cell Sci. 2000 111:1831-1839 and Morrissette and Sibley Microb. Mol. Bio. Rev. 2002 66:21-38).
In the case of hepatocyte invasion by sporozoites, injection of a very small number of Plasmodium sporozoites is sufficient to initiate infection, suggesting that the invasion process is extremely efficient. Furthermore, the process of hepatocyte invasion is extremely rapid, occurring within minutes after the infectious bite of the mosquito. The efficiency and rapidity of sporozoite invasion suggests that it involves specific interactions between parasite-encoded surface proteins and host molecules. Several lines of evidence suggest that the circumsporozoite protein (CS) plays a key role in this process (Stewart et al. Infect. Immunol. 1986 51:859-864; Stewart and Vanderberg J. Protozol. 1988 35:389-393; Menard, R. Cell Microb. 2001 3:63-73; Mota and Rodriquez Bioessays 2002 24:149-156). The CS protein of all Plasmodium species contains a highly conserved region, called region H, that is also found in the type I repeats of thrombospondin and some other adhesion molecules. It is believed that region H of CS binds to glycosaminoglycan chains of heparin sulfate proteoglycans (HSPGS) that are found on the surface of hepatocytes (Sinnis et al. J. Exp. Med. 1994 180:297-306). This binding is required for sporozoite attachment to hepatocytes, but is not necessary for invasion. Subsequent invasion with formation of a parasitophoros vacuole is tightly associated with exocytosis of the apical organelles. The attachment of the parasite to the host cell also triggers a transient cytosolic Ca2+ increase that is required for invasion. This increase in the Ca2+ concentration seems to induce the exocytosis of micronemes, a process required for the formation of the moving junction and the internalization of the parasite. Whether this increase in Ca2+ concentration is also required for activation of the actin-myosin based motility system, as seen with other non-Apicomplexan motility systems, is not known. The exocytosis of the apical organelles results in the release of the TRAP (Thrombospondin-related anonymous protein) molecule and its distribution along the surface of the sporozoite. TRAP is a transmembrane protein that contains two well-characterized adhesive modules, an A domain of Von Willebrand factor and a type 1 repeat of thrombospondin, in its extracellular domain. TRAP (−) parasites, created by gene-targeting technology in Plasmodium, are unable to invade mosquito salivary glands or infect liver cells in vivo after intravenous injection, suggesting that TRAP plays a key role in the invasion process (Sultan et al. Mol. Biochem. Parasitol. 1997 113:151-156). A structurally related molecule, Plasmodium CTRP, produced by the ookinete stage, probably plays a similar role as inactivation of CTRP in Plasmodium berghei and Plasmodium falciparum showed that CTRP is necessary for ookinete transformation into an oocyst, a stage of the life cycle that requires ookinete migration through the midget epithelium of the mosquito (Dessens et al. EMBO J. 1999 18:6221-6227; Yuda et al. J. Exp. Med. 1999 190:1711-1715; Templeton et al. Mol. Microb. 2000 36:1-9).
The merozoite form of the asexual life cycle in the blood stage attaches to the surface of the red blood cell (RBC) which initiates the invasion process of this host cell. Many of the surface proteins of the merozoite are thus exposed to the immune system and consequently are potential vaccine candidates. Many of these proteins are thought to play a role in merozoite invasion of RBCs but the details of their function and interactions remain unclear at best. Merozoite invasion takes place following initial interaction with the RBC surface followed by re-orientation to allow the apical end of the parasite to interact with the membrane of the host cell. This re-orientation allows the contents of the apical organelles to be released and a tight junction is formed between the merozoite surface and the RBC membrane. The tight junction moves along the surface of the merozoite, possibly via force generated by the actin-myosin motor, until the membrane fuses at the posterior end of the parasite, resulting in the formation of the parasitophorous vacuole contain the newly invaded parasite (Pinder et al. Parasit. Today 2000 16:240-245). While multiple proteins appear to be involved in this complex process of RBC invasion, very little is known about the particular role of any individual protein in the process. One of the surface proteins, merozoite surface protein I (MSPI) has been postulated to be involved in the initial interaction of the merozoite with the RBC surface (Holder and Freeman, J. Exp. Med. 1984 160:624-629; Holder et al. Nature 1985 317:270-273; Perkins and Rocco J. Immunol. 1988 141:3190-3196). The localization of this GPI-anchored protein is shared with a number of other GPI-linked proteins including MSP2, MSP4 and MSP5 (Smythe et al. Proc. Natl Acad. Sci. USA 1988 85:5195-5199; Marshall et al. Infect. Immun. 1997 65:4460-4467; Marshall et al. Mol. Biochem. Parasitol. 1998 94:13-25). Apical membrane antigen I (AMA I) is an integral membrane protein that is initially localized to the neck of the rhoptries although it re-distributes onto the surface of the merozoite (Deans et al. Mol. Biochem. Parasitol. 1984 11:189-204; Peterson et al. Mol. Cell Biol. 1989 9:3151-3154; Narum and Thomas Mol. Biochem. 1994 67:59-68; Marshall et al. Mol. Biochem. Parasitol. 1989 27:281-284; Peterson et al. Mol. Cell Biol. 1990 9:3151-3154). The function of AMAI is presently unknown. Within the rhoptries, a number of proteins have been identified that may be involved in the invasion process. These include the rhoptry-associated proteins 1 and 2 (RAP I and RAP2) (Ridley et al. Mol. Biochem. Parasitol. 1990 41:125-134; and Perrin et al. J. Clin. Invest. 1985 75:1718-1721). The soluble complex of these two molecules is delivered out to the rhoptries during invasion and is carried through into the parasitophorous vacuole (Baldi et al. EMBO J. 2000 19:1-9). The erythrocyte-binding antigen 175 (EBA 175) of Plasmodium falciparum is located in the micronemes and has been shown to bind to the RBC surface molecule glycophorin A in a sialic acid-dependent manner (Sim et al. Exp. Parasit. 1992 78:259-268; Wu et al. Proc. Natl Acad. Sci. USA 1996 93:1130-1134; Crabb et al. Cell 1997 89:287-296). Disruption of the gene encoding EBA175 to investigate the role of the conserved carboxy-terminal cysteine-rich domain, the transmembrane domain and the cytoplasmic domain suggested these regions were not essential for merozoite invasion (Reed et al. Proc. Natl Acad. Sci USA 2000 97:7509-7514). However, analysis of RBC invasion with these mutants suggested that the EBA175/glycophorinA pathway was disrupted. It appears that the mutant parasites now invaded using a sialic-acid independent pathway suggesting that the parasite has the ability to utilize alternative pathways for invasion of RBCs.
The involvement of an actin-myosin-based motor in invasion was suggested by Miller et al. based upon studies of the effect of cytochalasin B on Plasmodium invasion (Miller et al. J. Exp. Med. 1979 149:172-184). In Plasmodium, there are two genes for actin. Actin I is intronless and is expressed throughout the parasite life cycle, wherein actin II has an intron and is transcribed only in the sexual stages (Wesseling et al. Mol. Biochem. Parasitol. 1988 27:313-320; Wesseling et al. Mol. Biochem. Parasit. 1988 27:313-320, Wesseling et al. Mol. Biochem. Parasitol. 1989 35:167-176). The amino acid sequence of actin II diverges from previously characterized actins exhibiting only 79% sequence similarity to the sequence of actin I (Wesseling et al. Mol. Biochem. Parasit. 1988 27:313-320). The majority of actin in Apicomplexan parasites appears to be monomeric rather than being in the polymerized form. Localization studies using anti-actin antibodies have indicated that actin is present in the region between the plasma membrane and the inner membrane complex (IMC), which is composed of two closely aligned membranes. The involvement of myosin in Apicomplexan invasion was suggested by Dobrowski et al. and Pinder et al. based upon studies where invasion was reversibly inhibited by the myosin ATPase inhibitor butane-2,3-monoxime (Dobrowski et al. Mol. Microbiol. 1997 26:163-173; Pinder et al. J. Cell Sci. 1998 111:1831-1839). As in other species, Apicomplexa contains several myosin genes. Of these, MyoA has been disclosed as rather unique (Heintzehnan and Schwartzman J. Parasitol. 1997 87:429-432). This myosin is expressed in all Plasmodium invasive stages. The molecule is very small (approximately 90 kD) but the head domain displays the universally conserved ATP and actin binding sites. MyoA binds actin and actin is released in an ATP-dependent fashion. However, MyoA has several unique features, including virtually no recognizable neck domain (which is normally the binding site for a canonical myosin light chain) and a very short carboxy terminal tail. In the mature merozoite, MyoA is peripherally located with most staining occurring at the apical end and electron microscopy indicates that it is also located between the plasma membrane and the RAC (Pinder et al. J. Cell Sci. 1998 111:1831-1839).
Using a yeast two-hybrid system (Fields and Song Nature 1989 340:245-248; Luban and Goff Curr. Opin. Biotech. 1995 6:59-65), a molecule termed MTIP (MyoA Tail Interacting Protein) was isolated. This molecule binds to the short carboxy terminal tail of MyoA. Studies on the localization of this molecule have led to the proposal of a new model for the organization of the actin-myosin based machinery located between the plasma membrane and the IMC (Bergman et al. J. Cell Sci. 2003 116:39-49). It is believed that the “moving junction” which forms during invasion is a circumferential zone of attachment at the opening of the host cell invagination. This zone is characterized by a markedly thickened host cell membrane with increased electron density and is frequently accompanied by a constriction in the parasite body. The parasite enters the newly forming parasitophorous vacuole by capping the moving junction down its body. Eventually, the parasite becomes enclosed within a cavity delimited by the invaginated host cell membrane (Aikawa et al. J. Cell Bio. 1978 77:72-82; Michel et al. Int. J. Parasitol. 1980 10:309-313). The moving junction is a highly specialized interface of the parasite with the host cell, presumably utilizing cytoskeletal proteins, signaling molecules and receptors. However, very little is known about this interface. The protein MCP-I (Merozoite Capping Protein-1) was localized initially at the attachment site formed between the merozoite apical region and the erythrocyte (Klotz et al. Mol. Biochem. Parasitol. 1989 36:177-185). During the invasion process, MCP-1 migrates around merozoites in an anterior-to-posterior movement to finally persist at the posterior end of the newly invaded parasite (at the end nearest the erythrocyte membrane). MCP-1 is a 415 amino acid protein but interestingly is detected as a 60 kD protein in extracts from blood stage parasites. The amino terminal third of the molecule is an oxido-reductase domain but the function of this domain is not known. Thus, it appears that MCP-1 is localized to the moving junction in invading parasites but its role and potential interactions with other molecules remains unclear,
A new protein, Plasmodium falciparum Merozoite TRAP-like Invasin 1, referred to herein as PfMTI-1, has now been isolated and characterized. Blockage or inhibition of this protein is believed to prevent the malaria parasite from invading red blood cells, thus preventing or lessening the clinical severity of the disease. This protein and homologs or orthologs thereof serve as prime targets for immunologic and chemotherapeutic intervention.